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Preparation method of nucleic acid fingerprint database for detecting genital tract pathogens by MALDI TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry)

A fingerprint and pathogen technology, applied in the field of preparation of nucleic acid fingerprint library, can solve the problems of time-consuming and complicated operation

Active Publication Date: 2021-09-07
BIOYONG TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above methods inevitably have problems such as complicated operation and long time consumption.

Method used

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  • Preparation method of nucleic acid fingerprint database for detecting genital tract pathogens by MALDI TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry)
  • Preparation method of nucleic acid fingerprint database for detecting genital tract pathogens by MALDI TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry)
  • Preparation method of nucleic acid fingerprint database for detecting genital tract pathogens by MALDI TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] Embodiment 1, primer design

[0152] All detection target regions in the present invention are single-region detection, and the PCR primers correspond to the 3' ends of the above-mentioned reference PCR primers to ensure amplification. The properties were adjusted without affecting the specificity of the primers and without increasing the non-specificity of the primers.

[0153] Primers were designed for the target detection segments of 10 pathogens (the nuc segment of Staphylococcus aureus, the scpB segment of Group B Streptococcus, etc.), and after searching the NCBI database, the source of the target segment of each pathogen is as follows:

[0154] The target fragment of Staphylococcus aureus: the target fragment of Staphylococcus aureus is selected from the nuc fragment, > DQ399678.1:1-450 Staphylococcus aureus nuclease (nuc) gene, partial cds, its sequence number is DQ399678.1;

[0155] The target fragment of Group B Streptococcus: the target segment of Group B St...

Embodiment 2

[0172] Embodiment 2, build the detection mass spectrometry model of 10 kinds of pathogens

[0173] Using ABI 9700 PCR instrument, according to the instructions, the specific sites related to 10 kinds of reproductive tract pathogens were tested.

[0174] In this example, a multiplex primer system for detecting 10 kinds of reproductive tract pathogens was used to construct a mass spectrometry model for the detection of 10 kinds of pathogens. Set the blank control as deionized water, the negative control as human genomic DNA, and the positive control as the plasmid mixture of each target for detecting 10 kinds of reproductive tract pathogens.

[0175] The components used in the kit for PCR, PCR product purification and single base extension are:

[0176] serial number component name main ingredient Packing Specifications 1 Reaction solution I dNTPs, Tris-HCl, MgCl 2

323μL / tube×1 tube 2 Enzyme I Amplification enzyme, UNG enzyme 23μL / tube×1 ...

Embodiment 3

[0209] Embodiment three, isolate patient's pathogenic DNA

[0210] Sample source: vaginal secretions. Sampling personnel should wear clean hands and wear disposable gloves before sampling, and should avoid the menstrual period of the subject when collecting. The specimen collection and extraction methods are as follows: Cervical sampling swab: Rotate a plastic rod swab with a polypropylene fiber head and gently insert it into the vagina, stay for a while and then rotate clockwise 5 times. Take out the swab and dip it into a tube containing 3ml of sampling solution, discard the tail, and tighten the cap of the tube. The swab preservation solution should not be stored for more than one week at 2-8°C, and should not be stored for more than one month at -20°C. It can be transported in a curler with ice or a foam box with ice. It is recommended to use fresh swab preservation solution as much as possible.

[0211] Sample extraction: Bacterial DNA extraction kits were used to extra...

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Abstract

The invention provides a preparation method of a nucleic acid fingerprint database for detecting specific sites of eight genital tract pathogenic bacteria and two human papilloma virus types by a time-of-flight mass spectrometry. The genital tract pathogenic bacteria comprise staphylococcus aureus, group B streptococcus, neisseria gonorrhoeae, gardnerella vaginalis, candida albicans, mycoplasma genitalium, mycoplasma urealyticum, chlamydia trachomatis and virus types of HPV16 and HPV18 . The invention provides a novel method for detecting 10 genital tract pathogens by combining multiple PCR with clinical mass spectrometry for the first time, integrates multiple PCR, single base extension, mass spectrometry detection and other technologies, and establishes the preparation method of the nucleic acid fingerprint database, so that the model can amplify a detection template through the PCR technology, also can be used for detecting trace samples through the mass spectrometry technology, integrates the advantages of the two technologies, is far superior to a method for detecting a specific fragment of the pathogens by singly using PCR (Polymerase Chain Reaction), and has relatively high detection sensitivity.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and relates to a preparation method of a nucleic acid fingerprint library for rapidly identifying 10 reproductive tract pathogens by utilizing laser time-of-flight mass spectrometry technology. Background technique [0002] According to the latest survey report of the World Health Organization, for adult women, the most common disease is not a cold, but a genital tract infection, with a probability of more than 90%; the most common discomfort symptoms are not fever, cough, headache, etc., but It is genital itching, odor, pain, as well as frequent urination, urgency and so on. Moreover, the occurrence of many other diseases in women is also closely related to the dysfunction of the reproductive system, such as osteoporosis, coronary heart disease, diabetes, and chloasma. In a sense, the problem of the reproductive system is related to the health and happiness of a woman's life, and it c...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6895C12Q1/686C12Q1/6806C12Q1/14C12Q1/04C12N15/11
CPCC12Q1/708C12Q1/689C12Q1/6895C12Q1/686C12Q1/6806C12Q2600/16C12Q2600/166C12Q2537/143C12Q2533/101C12Q2565/627C12Q2545/113
Inventor 李维邬文燕张雪莲张海燕马庆伟
Owner BIOYONG TECH
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